Abstract

Background

The methylotrophic yeast, Hansenula
polymorpha is an industrially important microorganism, and
belongs to the best studied yeast species with well-developed tools for
molecular research. The complete genome sequence of the strain NCYC495 of
H. polymorpha is publicly available. Some
of the well-studied strains of H. polymorpha
are known to ferment glucose, cellobiose and xylose to ethanol at elevated
temperature (45 – 50°C) with ethanol yield from xylose significantly lower than
that from glucose and cellobiose. Increased yield of ethanol from xylose was
demonstrated following directed metabolic changes but, still the final ethanol
concentration achieved is well below what is considered feasible for economic
recovery by distillation.

Results

In this work, we describe the construction of strains of H. polymorpha with increased ethanol production
from xylose using an ethanol-non-utilizing strain
(2EthOH−) as the host. The transformants derived
from 2EthOH− overexpressing modified xylose reductase
(XYL1m) and native xylitol dehydrogenase
(XYL2) were isolated. These transformants
produced 1.5-fold more ethanol from xylose than the original host strain. The
additional overexpression of XYL3 gene coding
for xylulokinase, resulted in further 2.3-fold improvement in ethanol production
with no measurable xylitol formed during xylose fermentation. The best ethanol
producing strain obtained by metabolic engineering approaches was subjected to
selection for resistance to the known inhibitor of glycolysis, the anticancer
drug 3-bromopyruvate. The best mutant selected had an ethanol yield of 0.3 g/g
xylose and produced up to 9.8 g of ethanol/l during xylose alcoholic
fermentation at 45°C without correction for ethanol evaporation.

Conclusions

Our results indicate that xylose conversion to ethanol at elevated temperature
can be significantly improved in H.
polymorpha by combining methods of metabolic engineering and
classical selection.

Keywords

Background

The production of bioethanol from renewable feedstocks represents the largest
industrial fermentation process with over 85.2 billion liters of fuel ethanol
produced in 2012 [1]. Currently ethanol
is produced from traditional feedstocks (corn starch and sugarcane) and is known as
1st generation biofuel. Due to increased production
of ethanol and the uses of these feedstocks for food and feed applications, no
significant growth in 1st generation ethanol production
is possible. This is the main reason for continued interest in the development of
cost effective technology for the production of the 2nd
generation bioethanol from lignocellulosics. These feedstocks primarily consist of
commodity crop processing residues, field residues and wood waste processing
residues. Fast growing energy crops such as switch grass, miscanthus and trees that
can be cultivated in poor or marginal soils have also been touted as possible
sources of lignocellulosic feedstocks. In spite of the potential availability of
lower cost lignocellulosic feedstocks, the complex structure of these has hindered
the development of a cost effective technologies for 2nd
generation bioethanol production. The major problems which prevent large scale
bioethanol production from lignocellulosics are the absence of: environmentally
friendly and cheap technology for lignocellulose pretreatment and hydrolysis and the
unavailability of microbial strains which efficiently ferment the major pentose
sugars of hemicellulose, of which xylose is the most abundant sugar [2–4].
Some of the different approaches used to overcome these bottlenecks are the
development of Consolidated Bioprocessing (CBP) for the pretreatment, hydrolysis and
fermentation of lignocellulose to ethanol and the use of Simultaneous
Saccharification and Fermentation (SSF) process to ferment pretreated lignocellulose
[5,6]. In the last process, the pretreated lignocellulose is
subjected to enzymatic hydrolysis by cellulases and hemicellulases during the
fermentation which converts liberated sugars to ethanol and thus avoids end product
inhibition of the enzymes by these sugars. During SSF, microorganisms which
efficiently ferment pentose and hexose sugars are utilized [7]. As cellulases and hemicellulases express
maximal activities at temperatures in the range 50 – 60°C, it is desirable that the
microorganisms used for SSF have higher thermotolerance than the currently used
industrial ethanologens [8].

There are many promising ethanologenic microorganisms capable of fermenting the
major pentose sugar, xylose. The list includes natural xylose-fermenting yeasts,
such as Pichia (Scheffersomyces) stipitis, Candida shehatae,
Pachysolen tannophilus, Spathaspora
passalidarum, recombinant yeast Saccharomyces
cerevisiae, and several recombinant ethanologenic bacteria such as
Escherichia coli, Klebsiella oxytoca, Zymomonas
mobilis, several sp of Bacillus and
Lactobacillus [3,9–12]. Most of these ethanologens are mesophilic
organisms that cannot grow and ferment at temperatures above 40–42°C. A number of
ethanologenic high temperature anaerobic bacteria such as Thermoanaerobacterium saccharolyticum are promising organisms
capable of high-temperature xylose fermentation [13]. However, bacteria have some technological disadvantages when
compared to yeasts, such as susceptibility to bacteriophage lysis, uncertainty in
regulation that hinder the use of their biomass for feed, and some, unlike yeast,
have not been in use at industrial scale. With the exception of Kluyveromyces marxianus [14], Hansenula polymorpha, is
the most thermotolerant yeast known with growth up to a maximal temperature of 50°C
[15]. It has been demonstrated that
H. polymorpha can ferment glucose, cellobiose
and xylose [16] and is able to convert
glycerol to ethanol [17]. Wild-type
strains of this yeast normally ferment xylose up to a maximal temperature of 48°C,
whereas at 50°C fermentation is strongly suppressed. Genetic manipulation leading to
increase in intracellular trehalose following knock out of acid trehalase gene
ATH1 or the overexpression of the heat shock
proteins Hsp16 and Hsp104, have been demonstrated to allow normal xylose
fermentation at 50°C [18]. The ethanol
tolerance of H. polymorpha can be further
increased by the overexpression of the heterologous gene MPR1 [19] or endogenous
gene ETT1 [Ishchuk O, Abbas C, Sibirny A, in
preparation]. The methylotrophic yeast, H.
polymorpha is an industrially important microorganism, and belongs to
the best studied yeast species with well-developed tools for molecular research
[20,21]. The complete genome sequence of the strain NCYC495 of
H. polymorpha is publicly available
[22].

H. polymorpha can be a promising organism for
both CBP and SSF processes. Recombinant strains of this organism have been
engineered which express amylolytic and xylanolytic enzymes and directly ferment
starch and xylan to ethanol [23].
Considering SSF process, H. polymorpha belongs to
very few thermotolerant yeast species capable of xylose fermenting and could be the
organism of choice, however, it is not free from several drawbacks. Most
importantly, ethanol yield and productivity from xylose in wild-type strains of
H. polymorpha are very low. However, these
features could be improved by classical selection and metabolic engineering. Three
approaches have been used earlier for construction of H.
polymorpha strains which improved ethanol production from xylose. In
one line of investigation, H. polymorpha gene
XYL1 coding for xylose reductase (XR), two
paralogs of xylitol dehydrogenase (XDH) XYL2A and
XYL2B, were deleted in the strain CBS4732
with the expression of the bacterial gene xylA
from E. coli or Streptomyces coelicolor [24]. The corresponding transformants expressed xylose isomerase
activity and grew on xylose, but the amount of accumulated ethanol was very low
(both transformants and wild-type cells accumulated maximally 0.15 g of ethanol/l).
The overexpression of E. coli xylA together with
H. polymorpha XYL3 coding for xylulokinase
(XK), increased ethanol production, however, maximal ethanol accumulation did not
exceed 0.6 g/l at 48°C [25]. In the
second line of investigation, the H. polymorpha
XR gene was engineered by site specific mutagenesis to reduce affinity toward NADPH
using a similar approach developed for Candida
tenuis [26].
Consequently, genes coding for modified XR (XYL1m), native XDH (XYL2) and XK
(XYL3) were overexpressed in strain CBS4732
which yielded a 2-fold higher ethanol accumulation in corresponding transformants
reaching 1.3 g of ethanol/l [27]. In
third line of investigation, the wild-type H.
polymorpha strain NCYC495 (currently, the sequenced strain) was
selected as the initial host as it was shown to be a more efficient xylose fermenter
relative to the strain CBS4732. The H. polymorpha
mutant 2EthOH− unable to utilize ethanol as a sole carbon
source was isolated from strain NCYC495 and characterized by a 3-fold increase in
ethanol accumulation. Subsequently, the gene PDC1
coding for pyruvate decarboxylase (PDC) was cloned and overexpressed in strain
2EthOH−. The best selected transformants from
2EthOH− accumulated 2.5 g of ethanol/l at 48°C
[28]. This concentration is still
very low and has to be substantially increased before the strain can meet
requirements for industrial production using an SSF process.

In the current work, we describe the construction of more efficient H. polymorpha high-temperature ethanol producers from
xylose. For this, the combination of methods of metabolic engineering and classical
selection were applied. Strain 2EtOH− was used for
overexpression of H. polymopha genes XYL1m, XYL2, XYL3 and PDC1. The best selected transformant was used for the isolation of
mutants resistant to the anticancer drug 3-bromopyruvate which is known to inhibit
glycolysis [29–31]. The best mutant obtained showed a 15-fold
enhancement in ethanol synthesis from xylose when compared to the wild-type strain
accumulating up to 10 g/l of ethanol at 45°C.

Results

Construction of strains overexpressing engineered XR and native
XDH

Analysis of our previous data showed that the highest ethanol yield from
xylose was achieved using an ethanol-non-utilizing strain that was derived from
strain NCYC495. The corresponding mutant 2EthOH− was
characterized by a 3-fold higher ethanol yield on the third day of xylose
fermentation, relative to the wild-type strain. The underlying molecular nature
of the mutation in the strain 2EthOH− is not known as
can be caused by a change in one of the alcohol dehydrogenase isozymes
[28]. Recently, it was also
shown that overexpression of certain genes in H.
polymorpha led to an increase in ethanol production from xylose.
These changes may result from the cloning of an engineered XR and native XDH, XK
and PDC (XYL1m, XYL2, XYL3, PDC1)
[25,27,28]. We hypothesized that introduction of additional copies
of these genes into the genome of the mutant strain
2EthOH− could further improve ethanol production
during xylose fermentation. For the co-overexpression of genes XYL1m and XYL2
under control of the strong constitutive promoter of the gene GAP1 encoding glyceraldehyde-3-phosphate
dehydrogenase, the plasmid pX1m-Z-X2 was constructed and used to transform
strain 2EthOH−. Selection of transformants was
performed on media supplemented with increased concentration of the selective
agent zeocin assuming multicopy integration of the plasmid bearing the ZeoR selective marker gene. Transformants were selected on medium with
0.3 g of zeocin/l. After stabilization via cultivation in non-selective medium
for 10–12 generations, the cells were grown in a selective medium and subjected
to biochemical analyses. The best obtained transformant
2EthOH−/XYL1m/XYL2 was characterized by a
1.7-fold increase in specific activity of XR and a 10-fold higher activity of
XDH (Table 1). The overexpression of
XYL1m and XYL2 genes resulted in a 1.5-fold increase in ethanol production
during xylose fermentation in batch culture with limited aeration reaching
3.3 g/l of ethanol at 45°C with a 1.7-fold reduction in xylitol production when
compared to the parental strain (Table 2, Figure 1).

Selection of strains co-expressing XR and XDH in part with XK and/or
PDC

For further improvement of ethanol production from xylose the genes XYL3 or PDC1
were separately or in combination overexpressed in the background of the former
strain 2EthOH−/XYL1m/XYL2 using the expression
vectors pGLG61/XYL3, pGLG61 + prGAP + PDC1Hp
and pGLG61/XYL3/PDC1. The abovementioned plasmids were derived of pGLG61 and
contain a weakly expressed bacterial gene APH
encoding aminoglucoside-3-phosphotransferase as the dominant marker for
geneticin (G418) resistance, and the sequence of the HARS36 (TEL188) as autonomic
replicating sequence. Such elements ensure multiple tandem integration of pGLG61
into the H. polymorpha telomere regions on a
medium containing geneticin [32].
Transformants expressing XYL3 and both
XYL3 and PDC1 genes were selected on a medium supplemented with 0.5 g/l
geneticin while the strains expressing PDC1
were selected on a medium with up to 1.0 g/l of this antibiotic. All types of
transformants were stabilized. The presence of the corresponding plasmids in the
stabilized transformants was confirmed by diagnostic PCR.

The expression of XYL3 gene in the
background of 2EthOH−/XYL1m/XYL2 resulted in up to
2.6-fold increase in the specific activity of XK in selected transformant
2EthOH−/XYL1m/XYL2/XYL3 when compared to the
parental strain. The recombinant strain
2EthOH−/XYL1m/XYL2/XYL3/PDC1 co-expressing XYL3 and PDC1
genes was shown to have 1.8- and 7.6-fold enhancement of XK and PDC activity
when compared to the initial strain. The specific activity of the enzyme PDC in
strain 2EthOH−/XYL1m/XYL2/PDC1 was 18-fold increased
when compared to strain 2EthOH−/XYL1m/XYL2
(Table 1). Such remarkable increase
in PDC activity can only be explained by the multicopy integration of the
plasmid pGLG61 + prGAP + PDC1Hp into the
engineered strain 2EthOH−/XYL1m/XYL2/PDC1 obtained
following selection on the medium supplemented with 1 g of geneticin/l. All
other obtained strains harboring additional copies of XYL3 or/and PDC1 gene, were
characterized by identical specific activity of XR and XDH (Table 1). Results of ethanol production at the third
day of xylose fermentation at 45°C by the constructed strains are shown in
Table 2. The highest ethanol
production was observed for strain
2EthOH−/XYL1m/XYL2/XYL3. Such a strain showed a
2.3-fold improvement in ethanol production relative to the parental strain
2EthOH−/XYL1m/XYL2. Strains
2EthOH−/XYL1m/XYL2/PDC1 and
2EthOH−/XYL1m/XYL2/XYL3/PDC1, synthesized
approximately 1.5-fold higher amounts of ethanol than that of parental strain.
On the 3rd day of xylose fermentation, the best
selected strains expressing singly XYL3, PDC1
or a combination of these two genes in the background of strain
2EthOH−/XYL1m/XYL2, accumulated 7.44 g/l, 4.7 g/l
and 5.0 g/l of ethanol, respectively (Table 2). Thus, the expression of the PDC1 gene in the strain co-expressing all three genes XYL1m, XYL2, XYL3 involved in the initial stages of
xylose catabolism had no positive effect on ethanol production, though
overexpression of PDC1 alone in the
background of 2EthOH−/XYL1m/XYL2 strain is beneficial
for ethanol synthesis. The impact of XYL3
gene on ethanol production during xylose fermentation is more significant as
compared to PDC1 gene, as overexpression of
the XYL3 gene resulted in reduced xylitol
accumulation during alcoholic fermentation of xylose. The strain
2EthOH−/XYL1m/XYL2 produced 8.5 g of xylitol /l
with xylitol yield of 0.29 g/g from consumed xylose (Table 2, Figure 1). Expression of gene XYL3 resulted in a significant reduction of xylitol synthesis to
zero. By comparison, the overexpression of PDC1 had no influence on xylitol accumulation. The expression of
PDC1 in combination with XYL3, reduced xylitol yield to 0.04 g/g caused by
an increased activity of XK (Table 1).
The difference between xylitol production of strains
2EthOH−/XYL1m/XYL2/XYL3 and
2EthOH−/XYL1m/XYL2/XYL3/PDC1 can be explained by
the alteration in the specific activity of this enzyme. The activity of XK of
strain 2EthOH−/XYL1m/XYL2/XYL3 was 1.5-fold higher
than that of strain 2EthOH−/XYL1m/XYL2/XYL3/PDC1 and
this correlated with an increase in copy number of XYL3 gene in the genomes of both strains (Table 1). This demonstates that sufficient activity of
XK is necessary for the reduction of xylitol formation during xylose alcoholic
fermentation. If the level of XK is not high enough for complete direction of
xylulose to xylulose-5-phosphate, xylulose can be reduced back to xylitol via
the reverse reaction catalyzed by XDH [33,34].

Thus, the overexpression of three enzymes involved in the initial stages of
xylose catabolism, modified XR, native XDH and XK, in the background of an
ethanol-non-utilizing mutant 2EthOH−, significantly
improved ethanol yield from xylose (0.25 g/g xylose relative to 0.08 g/g xylose
in the mutant 2EthOH− after 3 days of xylose
fermentation at 45°C), ethanol specific production rate (0.059 g/g biomass/h
versus 0.017 g/g biomass/h for parental strain) and ethanol productivity
(0.14 g/l/h versus 0.04 g/l/h for initial strain). In spite of the improvements
made to xylose alcoholic fermentation, these are not adequate for a profitable
SSF process. Additional production of ethanol from xylose was attempted using
the best strain isolated via metabolic engineering approaches and subjected to
further strain selection by classical approaches.

Isolation and characterization of the ethanol overproducing strains
resistant to anticancer drug 3-bromopyruvate

3-Bromopyruvate (3-BrPA) has been known for some time as a promising
anticancer drug. It is well documented that this compound causes ATP depletion
which induce cell death as a result of blocking of glycolysis by the inhibition
of the glycolytic enzymes, hexokinase II, glyceraldehyde-3-phosphate
dehydrogenase and 3-phosphoglycerate kinase. In addition to glycolytic enzymes,
3-BrPA has been shown to have other targets of action [29]. We hypothesized that yeast mutants
resistant to 3-BrPA can have mutation in regulatory or structural glycolytic
genes leading to increased glycolytic flux and as a result elevated amount of
synthesized ethanol during fermentation. In our experiments, the best
ethanol-producing strain 2EthOH−/XYL1m/XYL2/XYL3
co-overexpressing XYL1m, XYL2 and XYL3 genes, was used as target for further
selection and improvement. Mutants resistant to 3-BrPA were isolated on solid
mineral medium containing xylose as the sole carbon source and supplemented with
0.11 mM 3-BrPA. Around 150 mutants resistant to 3-BrPA were selected and
evaluated for the ethanol production during xylose fermentation. The 110
selected mutants possessed increased ethanol production as compared to the
parental strain. In other words, approximately 70% of 3-BrPA-resistant mutants
isolated displayed increase in ethanol synthesis during xylose fermentation. The
average ethanol yield was increased on 10-20% as compared to the parental
strain. Biochemical characteristics and ethanol production of the best selected
mutant 2EthOH−/XYL1m/XYL2/XYL3/BrPA is presented in
Table 1. The
2EthOH−/XYL1m/XYL2/XYL3/BrPA mutant was
characterized by approximately 1.3-fold improvement in the ethanol yield as
compared to 2EthOH−/XYL1m/XYL2/XYL3 strain reaching
0.3 g/g xylose. This 3-BrPA-resistant mutant had no measurable changes in the
specific activities of XR, XDH and XK, as well as in xylitol production relative
to the parental strain 2EthOH−/XYL1m/XYL2/XYL3
(Table 1).

Representative profiles of ethanol synthesis, biomass accumulation, xylose
consumption and xylitol formation for the strains
2EthOH−,
2EthOH−/XYL1m/XYL2,
2EthOH−/XYL1m/XYL2/XYL3 and
2EthOH−/XYL1m/XYL2/XYL3/BrPA are shown in
Figure 1. The consumption of xylose
by constructed strains as well as by the initial parental
2EthOH− strain during fermentation was
incomplete, suggesting that xylose uptake could be a serious bottleneck leading
to inefficient xylose conversion to ethanol and this presents another potential
target for further improvement of ethanol production from xylose.

The best obtained 2EthOH−/XYL1m/XYL2/XYL3/BrPA
mutant accumulated 9.8 g/l of ethanol after 3 days of fermentation. The
synthesized ethanol concentration was corrected for evaporation at high
temperature xylose fermentation experiments. It was found that in model
experiments when ethanol at fixed concentrations was shaken in fermentation
medium under fermentation conditions, approximately 50% of it was evaporated at
45°C in 3 days of incubation (data not shown). Correction for ethanol
evaporation gives the calculated amounts of accumulated ethanol as approximately
20 g/l on the 3rd day of xylose fermentation at 45°C.
The strain 2EthOH−/XYL1m/XYL2/XYL3/BrPA, similarly to
the parental strain 2EthOH−/XYL1m/XYL2/XYL3, did not
accumulate xylitol in the medium.

Discussion

H. polymorpha belongs to the best studied yeast
species with well-developed tools for molecular research. It is used commercially
for production of recombinant vaccines, interferons, insulin, enzymes and other
products [21,22]. Its ability to ferment xylose to ethanol at
elevated temperature is known for 10 years; however, ethanol yield and productivity
are very low. At the same time, it rather efficiently converts glucose, cellobiose
and glycerol to ethanol [17,18]. Several
metabolic engineering approaches have been successfully developed to improve ethanol
production from xylose in H. polymorpha, however,
ethanol production remained quite low [24,25,27,28]. In the current work, we decided to combine several developed
earlier approaches of metabolic engineering with classical selection for ethanol
overproducing strain using selection for antimetabolite resistance. We demonstrated
that there is a positive cumulative effect for the overexpression of engineered XR
and native XDH and XK on ethanol production from xylose. Additional overexpression
of PDC1 gene coding for PDC did not lead to
further improvement of ethanol synthesis from xylose, though overexpression of
PDC1 in the background of XYL1m and XYL2
overexpressed strain increased ethanol production. The impact of XK on ethanol
production during xylose alcoholic fermentation is more pronounced when compared to
PDC assuming that PDC does not limit xylose conversion in strain expressing XR, XDH
and XK. Overexpression of XYL1m, XYL2 and
XYL3 in the background of non-identified
mutation in the strain 2EthOH−, led to substantial
increase in ethanol accumulation during xylose fermentation (7.44 g/l at 45°C
relative to 0.6 g/l in the wild-type strain NCYC495 and 2.05 g/l in the parental
strain 2EthOH−).

The method we used for selection of the ethanol-overproducers among
3-BrPA-resistant mutants has been, to our knowledge, demonstrated for the first time
to increase ethanol production in yeasts. It is interesting to note that 70% of
3-BrPA resistant mutants obtained in this work, produced more ethanol as compared to
the parental strain in xylose medium. High percentage of ethanol overproducers
provides support for the use of this method for positive selection of ethanol
overproducers. In mammalian cells 3-BrPA has multiple targets of action
[29–31]. It would be important to identify targets of action of this
inhibitor in yeast cells and gene(s) which mutations lead to 3-BrPA resistance and
ethanol overproduction. We also have found that selection of 3-BrPA-resistant
mutants can be successfully used for the isolation of ethanol-overproducing strains
in other yeast species, including S. cerevisiae
(unpublished observation). The exact molecular events underlying 3-BrPA resistance
in mutants of S. cerevisiae and H. polymorpha will be described from another separate
study [Hryniv, O., Dmytruk, K., Sibirny, A., in preparation].

Thus, in this work we were able to increase accumulation of ethanol from xylose to
10 g/l relative to 0.6 g/l in the wild-type strain, or more than 15 times by
combining the approaches of metabolic engineering and classical selection. The
maximal observed level of ethanol produced from xylose by the best isolated strains
(near 10 g/l at 45°C results in an ethanol yield of 0.3 g/g from xylose). These
results make H. polymorpha close to known
promising organisms for the use in SSF process. Still this ethanol concentration is
lower than ethanol production in mesophilic xylose fermenting organisms such as
P. stipitis (0.35-0.44 g/g xylose) and
S. passalidarum (0.42 g/g xylose), but
similar to the best engineered strain of thermotolerant yeast K. marxianus (0.31 g/g xylose under anaerobic
conditions at 45°C) [12,35,36]. However, the ethanol productivity in the best H. polymorpha isolated strain is much higher when
compared to the best engineered strain of K.
marxianus (0.179 g/l/h versus 0.054 g/l/h at 45°C). To be
industrially feasible, ethanol yield in H.
polymorpha has to be further increased to be close to the theoretical
maximum. We are currently using other targets for metabolic engineering (e.g. xylose
transport, pentose phosphate pathway). This we hope can lead to further increase in
ethanol yield from xylose during high-temperature alcoholic fermentation of this
promising organism.

Conclusions

Methods of metabolic engineering and classical selection were successfully applied
for construction of more efficient H. polymorpha
ethanol producers from xylose, leading to 15-fold enhancement in ethanol synthesis
from xylose as compared to the wild-type strain.

Molecular-biology techniques

Standard cloning techniques were used as described [37]. Genomic DNA of H. polymorpha was isolated using the Wizard® Genomic DNA
Purification Kit (Promega, Madison, WI, USA). Restriction endonucleases and DNA
ligase (Fermentas, Vilnius, Lithuania) were used according to the manufacturer
specifications. Plasmid isolation from E.
coli was performed with the Wizard® Plus SV Minipreps DNA Purification System (Promega, Madison, WI,
USA). DNA fragments were separated on a 0.8% agarose (Fisher Scientific, Fair
Lawn, NJ, USA) gel. Isolation of fragments from the gel was carried out with a
DNA Gel Extraction Kit (Millipore, Bedford, MA, USA). PCR-amplification of the
fragments of interest was done with Platinum® Taq DNA Polymerase High Fidelity (Invitrogen, Carlsbad, CA, USA)
according to the manufacturer specification. PCRs were performed in GeneAmp® PCR
System 9700 thermocycler (Applied Biosystems, Foster City, CA, USA).
Transformation of the yeast H. polymorpha by
electroporation was carried out as described previously [38].

Plasmid construction

Plasmid pX1m-Z-X2 was used for overexpression of modified XYL1m gene and native XYL2 gene [27].
Plasmids pGLG61/HpXYL3 [25] and
pGLG61 + prGAP + PDC1Hp [26] were used for overexpression of
XYL3 and PDC1 genes, respectively. For
simultaneous overexpression of XYL3 and
PDC1 genes a NarI-restriction fragment
containing GAPp-PDC1-AOXt was isolated from
the plasmid pGLG61 + prGAP + PDC1Hp and
cloned into the NarI-linearized and dephosphorylated vector pGLG61/HpXYL3. The
resulting plasmid was named pGLG61/XYL3/PDC1 (Figure 2).

Biochemical methods

The specific activities of XR, XDH and XK in cell extracts were determined
spectrophotometrically as described before [27].

The PDC activity in cell extracts was determined spectrophotometrically
according to the methods described earlier [39]. Samples for enzyme activity measurements were taken from
the cultures on the third day of xylose fermentation at 45°C. The enzyme
activity was measured directly after the preparation of cell-free extracts as
described before [28].

All assay experiments were repeated at least twice.

Selection of 3-bromopyruvate (3-BrPA) resistant mutants

For isolation of 3-BrPA-resistant mutants solid mineral medium containing
glucose or xylose as sole carbon sources with 0.11 mM of selective agent was
used. Cell suspension of 2EthOH−/XYL1m/XYL2/XYL3
strain was plated on the 3-BrPA containing medium for final
OD600 = 0.1/ml and incubated at 37°C for five days.
Single 3-BrPA-resistant colonies were picked up, re-streaked on fresh YNB plates
containing 0.11 mM of 3-BrPA and used for fermentation experiments. Selected
mutants were stable and possessed resistance to the selective agent even after
6 month growth on agar slant cultures in YPD medium with regular transfer to
fresh YPD medium every month.

Analyses

Cells of transformants were grown in 100 ml of YPX medium (10 g/l yeast
extract, 20 g/l peptone, 40 g/l xylose) in Erlenmeyer flasks (bottle size -
300 ml) for 2 days and then inoculated into the 40 ml of YNB medium with 90 g/l
xylose in 100 ml Erlenmeyer flasks. Fermentation was carried out at a
temperature of 45°C with limited aeration (140 revolutions/min). Concentrations
of xylose, xylitol and ethanol from fermentation in medium broth were analyzed
by HPLC (PerkinElmer, Series 2000, USA) with an Aminex HPX-87H ion-exchange
column (Bio-Rad, Hercules, USA). A mobile phase of 4 mM
H2SO4 was used at a flow rate
0.6 ml/min and the column temperature was 35°C. Alternatively concentrations of
ethanol in the medium were determined using alcohol oxidase/peroxidase-based
enzymatic kit “Alcotest” [40]. For
high temperature xylose fermentation experiments, the ethanol concentration was
corrected for evaporation. The evaporation coefficient was calculated as a
decrease of known concentration of ethanol at 45°C temperature.

Experiments were performed at least twice.

Abbreviations

SSF:

Simultaneous saccharification and Fermentation

CBP:

Consolidated bioprocessing

XR:

Xylose reductase

XDH:

Xylitol dehydrogenase

XK:

Xylulokinase

PDC:

Pyruvate decarboxylase

3-BrPA:

3-Bromopyruvate

Declarations

Acknowledgements

This work was supported by Science and Technology Center in Ukraine (Grant
No. 5505) and National Academy of Science of Ukraine (Grant No. 5-14).

Competing interests

The authors declare that they have no competing interest.

Authors’ contributions

OOK and JR carried out strains construction, evaluation of enzymes activity,
fermentation experiments and co-drafted the manuscript. OBH participated in cloning
and strain construction. CAA commented and approved the manuscript. KVD participated
in design of cloning and strain construction, analyzed the date and co-drafted the
manuscript. AAS provided guidance and suggestions for experimental design, wrote and
edited the manuscript. All authors participated in finalizing the manuscript read
and approved the final version of the manuscript.

Authors’ Affiliations

(1)

Department of Molecular Genetics and Biotechnology, Institute of Cell
Biology, National Academy of Sciences of Ukraine, Lviv, Ukraine

(2)

Department of Biotechnology and Microbiology, University of Rzeszow, Rzeszow, Poland

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